Film based addressable programmable electronic matrix articles and methods of manufacturing and using the same

ABSTRACT

An electronic device adapted for performing molecular biological processes. The device includes a flexible polymeric substrate having a first surface and a second surface. A plurality of microlocations interrupt the first surface, and each of said microlocations include an electrode disposed on the second surface of the flexible substrate. A hydrophilic matrix is positioned on the first surface of the flexible substrate and is capable of electrical contact with the electrode.

FIELD OF THE INVENTION

This invention relates to film based addressable programmable electronicmatrix articles and methods for manufacture and use of the same. Thearticles are manufactured on a flexible film, and are suited for usewith patterned polymer films tailored to selectively bind or react withvarious target species, including biologically active molecules.

BACKGROUND OF THE INVENTION

One of the most important activities of modern medical and biochemicalfields is conducting medical diagnostic assays, such as cell cultureassays, immunoassays, DNA hybridization assays, robot assisted samplehandling processes, and microfluid sample processing. These activitiespermit safe and effective medical diagnoses, as well as thorough andaccurate biochemical investigations and research.

Automated microbial culturing systems have been developed in recentyears to test for a variety of diseases in a clinical laboratorysetting. These systems often include culture tubes containing samples,selective growth media, and a fluorescent indicator that responds to thegrowth of the microorganisms. The tubes are continually processed by anoptical reader that measures changes in fluorescent properties of thesample in order to detect such microbes as tuberculosis or antibioticresistant Staph. aureus. Unfortunately, such systems can take days toculture a sufficient quantity of the microbes necessary foridentification with standard test methods.

Biocards. such as those described in U.S. Pat. No. 5,609,828, have alsobeen developed to carry out multiple assays from a single sampleextracted from blood, fluids, or other tissue of a patient. Thesesamples are usually examined using spectroscopic or other automatedanalysis techniques. Biocards are typically molded in plastic and aredesigned to receive a liquid sample into a series of small sample wellsformed in the card. Each sample well normally contains a different setof dried reagent (selective growth nutrients and indicator dies) foridentifying different biological agents within the sample. Duringanalysis, the sample enters an intake port, collects in an intakereservoir, and travels along distribution channels to the sample wells.Each sample well also typically includes a bubble trap designed to trapgases formed by growing microorganism colonies. The reagents within thesample wells dissolve when the fluid sample is introduced. Afterincubation of the sample in the sample wells, a card reader performsautomated spectroscopic or fluorescence analysis on each well. Althoughanalysis with biocards can be successful, analysis times are quite longunless the microorganisms are first cultured to increase their number.In addition, closely related strains of microorganisms are hard todifferentiate by these methods.

Efforts have been made to develop assay techniques for the analysis ofnucleic acids and proteins that shorten the delay associated withculture techniques, increase the specificity of the assays, and providemeans for detecting new diseases. One such effort has been thedevelopment of DNA amplification technologies that provide a means toproduce hundreds of millions of copies of a selected DNA target in lessthan one hour. Microorganisms of interest are first lysed to releasetheir DNA material. The DNA material is isolated and then treated withreagents to perform an amplification of an oligonucleotide sequencespecific to the microorganism of interest. While polymerase chainreaction (PCR) is the most well known of these amplification methods, itrequires temperature cycling and continued reagent additions. Othermethods, such as a strand displacement amplification approach developedat Becton Dickinson of Franklin Lakes, N.J., can be performed in asingle sample well in 15 minutes at constant temperature. Suchamplification methods help to overcome problems related to complexityand sensitivity in genomic DNA analysis.

Once the DNA target has been “amplified” by reproduction to producenumerous amplicons, complementary oligonucleotide probes can be used tocapture the DNA amplicons. These probes selectively retain the DNAamplicon, allowing them to be isolated and identified. However, theseprobes have traditionally relied upon diffusion controlled processes tocapture the DNA amplicons. Diffusion can take hours to complete, and isa significant hurdle to rapid identification of the DNA target. Althoughsuch diffusion methods are significantly quicker than prior cell culturetechniques, they are still relatively slow compared to the rapid rate ofDNA amplification.

An alternative to DNA amplification, known as the Southern Blot,involves cleaving the DNA with restrictive enzymes, separating the DNAfragments on an electrophoresis gel, blotting to a membrane filter, andthen hybridizing the blot with specific DNA probe sequences. Thisprocedure effectively reduces the complexity of the crude DNA sample,thereby helping to improve the hybridization specificity andsensitivity. However, the total number of targets generated in aSouthern Blot is far less than the number of amplicons generated by DNAamplification methods. In addition, the electrophoretic separation cantake hours to complete.

Recently, efforts have been made to hasten the separation and capture ofDNA amplicons. Researchers have developed micro-electrode arrays thatspeed up the capture process by using free-field electrophoresis toconcentrate and purify target DNA on the individual probes of the array.These arrays create a charged electrical field that isolates the chargedDNA amplicons at one or more probes on the array. This type ofmicroelectronic array, known as an “addressable programmable electronicmatrix” (“APEX”), can reduce the time to perform the capture processfrom hours to minutes. A number of patents describe silicon based chipshaving APEX arrays. For example, U. S. Pat. Nos. 5,653,939 and 5,632,957teach the manufacture-of rigid silicon APEX arrays using a lithographicprocess. Although these silicon APEX arrays permit enhanced capturerates, they are relatively expensive to produce. Also, they cannot beeasily reused without being cleaned and repatterned with DNA probes in amanufacturing environment. Even though they are relatively expensive,the silicon APEX arrays are typically used once and then discarded.

Thus, existing APEX arrays have improved the speed of performing DNAidentification tests, but have failed to address the need forcost-effective, mass manufacturable APEX assay systems. In view of thesignificant expense associated with existing APEX chip systems, a needexists for an APEX chip that provides the increased speed of aprogrammable microelectronic matrix, but can be done for less expensethan existing silicon-based chips.

SUMMARY OF THE INVENTION

The present invention is directed to a film based microelectrode arraydevice adapted for processing of chemical, biological, or particulatematerials at electronically addressed micro-locations. Uses for thedevice include chemical- and molecular biological-type analyses,including nucleic acid hybridization reactions, antibody/antigenreactions, various cell sorting operations, and synthesis reactionsincluding DNA amplifications and various free-field electrophoresismanipulations.

In specific implementations, the device includes a flexible polymericsubstrate having a first or upper surface and a second or lower surface.A plurality of microlocations interrupts the first surface, and each ofthese microlocations preferably includes an electrode disposed on thefirst or second surface of the flexible substrate. Circuit tracesconnect the electrodes to larger contact pads also disposed on the firstor second surface of the flexible substrate. A hydrophilic permeationmatrix and optionally a biologically receptive polymer is positioned onthe first surface of the flexible substrate and is capable of supportingelectrical contact between the electrode and a fluid sample placed incontact with the first surface of the flexible substrate. Flexiblepolymeric substrates of the present invention are particularly wellsuited for the manufacture of APEX chips and enable the manufacture ofnovel APEX biocards, and APEX spools.

In certain implementations of the invention, the flexible polymericsubstrate is formed of polyimide. The flexible polymeric substrate maybe completely, substantially, or partially formed of the polyimide, andmay be combined with other materials. It will be appreciated that theinvention is also directed to any flexible substrate that permits theformation of a first surface and a second surface, with electrodesinterrupting the first surface. The substrate and electrodes should alsopermit the retention of a hydrophilic permeation matrix, such as aderivitizable gel, in a manner such that programmable free-fieldelectrophoresis processes can be conducted using the substrate andelectrodes. The hydrophilic permeation matrix may include a biologicallyreceptive gel, or may have inherent biological receptive properties.

In one implementation, the plurality of microlocations interrupting thefirst surface includes vias that extend from the first surface into thepolymeric substrate. The vias provide a location for the formation ofthe electrodes. The electrodes are formed, for example, by securing aconducting metallic layer on the second or lower surface of the flexiblesubstrate and then selectively removing the polymeric substrate abovethe conducting metallic layer. The polymeric substrate is selectivelyremoved to expose the conducting metallic layer, thereby formingelectrodes. Removal of the substrate is performed by chemical etchingmethods, plasma removal methods, laser removal methods, or othersuitable methods.

In other examples, the electrodes are formed by depositing a metalliclayer on the first or upper surface of the flexible polymeric sheet.After the metallic layer is deposited, a non-conductive masking layer isapplied over the metallic layer. This masking layer is selectivelyremoved by an appropriate methodology, such as photo-lithography, inorder to expose portions of the metallic layer to form a plurality ofexposed electrodes.

It will also be appreciated that in certain implementations of theinvention the electrodes formed by exposure of the metal layer maysubsequently be enlarged by deposition of additional metal on theelectrode. The deposition of additional metal is accomplished, forexample, by electroplating the electrodes to selectively thicken them.Alternatively, small metallic pieces may be mechanically inserted intoeach via above each electrode, and these metallic pieces can besubsequently fused by heat or pressure in order to thicken the electrodeat the microlocation. Such thickening of the electrode can enhanceperformance of the APEX by allowing tailoring of the contact surface,e.g., by selection of electrode metals such as gold. Also, theadditional metal can enhance performance by effectively securing themetal layer to the substrate by fusing the electrode within the via.

The electrodes formed by exposure to the metal layer may be connected bymetal traces to much larger contact pads located elsewhere on the firstor second surface of the flexible polymeric substrate. When theelectrodes are connected by vias to the circuit traces and contact padsprinted on the second surface of the substrate, the first surfacebearing the exposed electrodes can be directly laminated to a fluidhandling architecture that directs the fluid sample to the electrodearray. The contact pads on the second surface can be designed to extendto the edge of the device and mate directly with a voltage control unitby sliding the chip into a mated connector. This design overcomesarduous wire bonding processes of prior art APEX chips and overcomes theneed to encapsulate the lead wires in a protective material, since theyare shielded by the flexible polymeric substrate from exposure to thefluid sample.

The electrodes may be recessed within the vias in the flexible polymericsubstrate and routed through conducting traces on the second surface ofthe flexible polymer substrate. Such a one-piece construction comprisesa microwell above the electrode and provides protection for the circuittraces without the need for an additional protective layer. Themicrowell can serve as a reservoir for a sample or it can be partiallyor completely filled with a hydrophilic polymer to introduce apermeation layer or a biologically receptive gel above the electrode. Inthis manner the improved APEX device of the present invention overcomesthe difficulties in mating a circuit board to a microwell array, asdescribed in U.S. Pat. No. 5,605,662.

A hydrophilic permeation matrix on the top surface of the electrodespermits free-field electrophoresis of samples placed on the top surfaceof the APEX circuit while, at the same time, impeding the diffusion oflarge biomolecules, biologically receptive molecules, biologicallyreactive molecules, reagents or products through the matrix to thesurface of the electrode. This matrix may be biologically receptive andused to attach biomolecules to the electrode. In certainimplementations, a separate biologically receptive gel may be chemicallyor physically adhered to the permeation matrix or the electrode itselfto support the attachment of biomolecules. The biologically receptivegel or permeation matrix may include an azlactone-functional monomer. Asused in the present invention, “azlactone-functional monomer” means amonomer whose structure includes an azlactone moiety that optionally hasbeen bound to a biomolecule by a ring opening reaction of the azlactoneto form, e.g., an amide bond. The gel is preferably swellable, therebyproviding an increased concentration of biologically receptive moleculesper unit area when used as a coating. The biologically receptive gel maybe configured and arranged such that it may be patterned to a highresolution. This gel may be cured by actinic radiation, and the curedcomposition may be capable of reacting with selective biomolecules toimmobilize the biomolecules immediately above the micro-electrodes.

An azlactone-functional gel or permeation matrix can be employed andlocalized to the region just above the electrodes and within theconfines of vias, in a specific embodiment of the invention. In thisembodiment, some or all of the azlactone groups over a particularelectrode can be reacted, through simple addition reactions, withselected amine- or thio-terminated biomolecules to immobilize thesebiomolecules above the electrode, thus forming a biologically receptivegel. These selected biomolecules may be oligonucleotide or antibodyprobes, enzymes such as polymerase, or other biomolecules useful foranalysis. One advantage of the azlactone based APEX films of the presentinvention is that the azlactone and the biomolecules can be anchored tothe microlocations of the film by simple web coating, inkjet printing,or thermal imaging technologies, without the need for reagent additionsor product removal, thereby greatly simplifying their manufacturerelative to prior art APEX chips.

As used in the present invention, “hydrophilic permeation matrix” is apolymeric material capable of swelling in water, such as by adsorptionor chemical interaction, and refers to the matrix material either beforeor after swelling in water. “Photocrosslinker” means a chemical speciesthat is capable of binding two or more polymer molecules in response tothe application of electromagnetic radiation. The photocrosslinker iscapable of attaching to the polymer molecules at a site other than theend of a growing polymer chain. “Copolymeric crosslinker” means achemical species that is capable of binding two or more polymermolecules, and that attaches to a polymer at the end of a growingpolymer chain.

As used herein, “biologically active” includes biochemically,immunochemically, physiologically. or pharmaceutically active; and“biologically active molecule” and “biomolecule” are usedinterchangeably and include antibodies, antigens, enzymes, cofactors,inhibitors, hormones, receptors, coagulation factors, amino acids,histories, vitamins, drugs, cell surface markers, carbohydrates,proteins and polypeptides, DNA (including DNA oligonucleotides), RNA(including RNA oligonucleotides), and derivatives of the foregoing.“Substituted” means substituted by conventional substituents which donot interfere with the desired product, e.g., substituents can be alkyl,alkoxy, aryl, phenyl, halo (F, Cl, Br, I), cyano, nitro, etc.

The invention provides a flexible polymeric substrate that can becontinuously produced on a commercial scale in the convenient form of aroll-good that can be readily stored and handled. The finished roll-goodcan be used directly after application of the hydrophilic matrix orbiologically active gel to perform electrophoresis assisted processingof chemical, biological, or particulate materials at electronicallyaddressed micro-locations. For example, the flexible polymeric substratecan be used on a spool or roll-good in a continuous reel-to-reel processin which a plurality of addressable programmable electrode matrices aresequentially supplied, used, and taken up. Alternatively, the roll-goodcan be cut into sections containing a plurality of APEX arrays forincorporation into biocards. Also, alternatively, the roll-good may becut into separate APEX units for individual use. It will be appreciatedthat the flexible polymeric substrate containing the APEX unit or unitscan further include or be combined with other microelectronic,microoptical, microstructural, and/or micromechanical elements. Thesemicroelements may be incorporated into multilayer articles.

The invention is further directed to a multi-sample APEX processingspool and system in which the first surface of a roll of the APEX filmmay be laminated to a flexible plastic fluid handling architecture, saidfluid handling architecture designed to direct 2-10,000 independentbiological samples to the corresponding number of independentlyaddressable APEX arrays on the APEX film for processing. The spool canbe advanced through a machine that infects a sample into a subset of theAPEX arrays for processing. Once the samples are assayed, the spool canbe advanced, exposing additional APEX arrays. A voltage control unitsimultaneously can provide processing currents or voltages to each ofthe APEX arrays on the spool at any one time. A detection system canprovide optical, electrical, or mechanical signals in response to thebiological events at the individual electrodes of each APEX array. Thus,continuous automated processing, whether sequentially or simultaneously,of thousands of samples can be carried out using inexpensive, disposablearrays.

The invention is further directed to a multi-sample APEX biocard andsystem in which a sheet of APEX film may be laminated to a semi-rigidglass or plastic fluid handling architecture, said fluid handlingarchitecture designed to direct numerous independent biological samplesto the corresponding number of independently addressable APEX arrays onthe APEX film for processing, preferably between 2 and 200 samples. Thefluid handling architecture can be anything from simple barriers betweenAPEX arrays, including open wells into which sample is injected when thecassette is horizontal, to closed channel structures into which a sampleis injected with a syringe or pump. A machine can be adapted to acceptand operate the APEX cassette. The machine may comprise a sampleinjection unit to provide numerous (preferably from 2 to 200)independent biological samples onto the biocard via ports in the fluidhandling architecture; a voltage control unit that can simultaneouslyprovide processing currents or voltages to each of the APEX arrays; anda detection system that can provide optical, electrical, or mechanicalsignals in response to biological events at the individual electrodes ofeach APEX array.

The invention is further directed to a method of performing molecularbiological processes, the method including providing an electronicdevice containing a flexible polymeric substrate having a first surfaceand a second surface. The electronic device may comprise an array ofelectrodes disposed on the first or second surfaces of the flexiblesubstrate and exposed toward the first surface. A hydrophilic permeationmatrix and biologically receptive polymer having a covalently anchoredbiological molecule can be positioned on the first surface of theflexible substrate such that they are in contact with the array ofelectrodes, after which an electrical force can be applied to theelectrodes so as to effect electrophorcsis-assisted processing of thebiological sample. By similar means, electrophoretic processing ofchemical or particulate matter also can be envisioned.

In specific implementations of the method of the invention, theelectronic device may include a plurality of electrodes. The chargepotential of the electrodes preferably can be individually controllable.Alternatively, the charge potential of the electrodes can be controlledtogether as one unit, or as a group of sub-units, each sub-unit having aplurality of electrodes electronically coupled to one another. Duringoperation, the charge potential of the electrodes optionally can bealtered in order to modify the electrical field of the electronicdevice. Such alterations to the electrical field can be used, forexample, to first attract all the charged biologically active moleculesto the biologically receptive gel, and then to subsequently repel thebiologically active molecules that are not retained by the receptivegel. These efforts can result in accumulation of desired biomoleculesand removal of undesired molecules from the positions of the electrodesbased on specific biomolecular recognition by anti-body oroligonucleotide probes associated with the individual micro-electrodes.

Other features and advantages of the invention will be apparent from thefollowing detailed description of the invention and the claims. Theabove summary of principles of the disclosure is not intended todescribe each illustrated embodiment or every implementation of thepresent disclosure. The figures and the detailed description that followmore particularly exemplify certain embodiments utilizing the principlesdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Principles of the invention may be more completely understood inconsideration of the detailed description of various embodiments of theinvention that follows in connection with the accompanying drawings inwhich:

FIG. 1 is a bottom plan view of an APEX circuit constructed inaccordance with the present invention.

FIG. 2A is an enlarged top view of a flexible polymeric substratecontaining an APEX circuit constructed in accordance with the presentinvention, showing microwell locations.

FIG. 2B is an enlarged bottom view of the flexible polymeric substratedepicted in FIG. 1, showing the bottom of the APEX circuit constructedin accordance with the present invention, including electrical contactpoints.

FIG. 3 is a fragmentary cross-sectional view of a flexible polymericsubstrate and an electrode constructed in accordance with animplementation of the invention.

FIGS. 4A, 4B, 4C, 4D and 4E are fragmentary cross-sectional views of anAPEX circuit manufactured on a flexible polymeric substrate, showing theAPEX circuit during various steps of manufacture.

FIG. 5 is a partial cross-sectional view of a flexible polymericsubstrate and electrode constructed in accordance with anotherimplementation of the invention.

FIG. 6 is a partial cross-sectional view of a flexible polymericsubstrate and electrode constructed in accordance with anotherimplementation of the invention.

FIG. 7 is a partial cross-sectional view of a flexible polymericsubstrate and electrode constructed in accordance with anotherimplementation of the invention.

FIGS. 8A, 8B, 8C, 8D, and 8E are partial cross-sectional views of anAPEX circuit manufactured on a flexible polymeric substrate, showing theAPEX circuit during various steps of manufacture.

FIG. 9 is a cross-section view of an APEX circuit including afluid-handling architecture in accordance with another implementation ofthe invention.

FIG. 10A and 10B is are perspective views of a biocard in accordancewith an implementation of the invention.

FIG. 11 is a schematic representation of the roll-to-roll process of theinvention.

While principles of the invention are amenable to various modificationsand alternative forms, specifics thereof have been shown by way ofexample in the drawings and will be described in detail. It should beunderstood, however, that the intention is not to limit the invention tothe particular embodiments described. On the contrary, the intention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an electronic device adapted forprocessing chemical, biological, or particulate materials atelectronically addressed microlocations. In specific implementations,the device includes a flexible polymeric substrate having a firstsurface and a second surface. The first surface is preferably orientedupward when in use, and the second surface is preferably orienteddownward. A plurality of microlocations interrupts the first surface,and each of these microlocations preferably includes an electrodedisposed on the first or second surfaces of the flexible substrate.

The flexible polymeric substrate allows the electronic device to bemanufactured in a continuous, roll-to-roll, mass-produced manner, freeof the inherent limitations experienced by electronic devicesmanufactured batch-wise on silicon substrates. In addition, the flexiblepolymeric substrate allows the electronic device to be manufactured andstored as a spool or roll-good. This roll-good can contain a pluralityof independent electronic devices that are separated from one anotherduring use or which are used sequentially while on the roll.

A hydrophilic permeation matrix and optionally a biologically receptivegel is positioned on the first surface of the flexible substrate and iscapable of electrical contact with the individual electrodes. Inparticular embodiments, the permeation matrix and biologically receptivegel are localized on the individual microlocations. The permeationmatrix permits free-field electrophoresis of macromolecules andparticles while preferably impeding their diffusion to the surface ofthe electrode, where they might foul the electrode or undergoelectrochemical reactions. The biologically receptive gel permits thecovalent binding of biomolecules such as DNA probes, antibodies,enzymes, and the like, useful for processing of chemical, biological, orparticulate materials near the electrode surface. In one implementationof the invention, the matrix and gel permit the isolation of biologicaltargets, such as DNA fragments, antigens, and other biologically activemolecules.

During operation of the invention in certain embodiments, a smallquantity of test liquid containing a mixture of biological targetspecies is applied to the APEX array. A biasing signal (voltage orcurrent) is applied to selected electrodes, thereby acceleratingtransport of the target species into the hydrophilic matrix above theselected electrodes. The biasing voltage is subsequently stopped, withthe target species concentrated at one or more of the microlocations. Ifbiomolecular probes are present at the microlocations, the selected testspecies specifically hybridize or otherwise interact with thebiomolecular probes, while other test species not specificallyrecognized by the probes are free to diffuse back into solution.Optionally, a reverse voltage bias is applied to the electrodes toquickly remove all unhybridized species from the microlocations.

This process can be repeated at different microlocations, such that atest species is moved from one microlocation to another until it reachesthe microlocation having a probe specific to that species, and becomesbound there. The presence or absence of the target species is thendetermined using conventional identification methods, such as measuringthe fluorescent signature for a reporter species attached to theselected target species at each microlocation.

In other embodiments, the microlocations can be configured to serve assites for chemical reactions. For example, an enzyme can be covalentlyanchored at a microlocation. A biasing signal can be applied to thatmicrolocation to direct an enzyme substrate to that microlocation, wherethe enzyme can act upon the substrate. By reversing the bias, productsof the enzymatic reaction can be driven away from the electrode, orredirected to a different electrode based on free field electrophoretictransport.

To more fully illustrate the invention, reference is now made to thefigures, which show various implementations of electronic circuitsconstructed in accordance with the present invention. FIG. 1 is a bottomplan view of an addressable programmable electrode matrix (APEX) circuit10 constructed on flexible polymeric substrate 12. Circuit 10 includesnumerous microelectrodes that are connected by metal traces 20 toindividually addressable contact pads 21, which may in turn be connectedto current or voltage control circuitry (not shown). In this embodiment,microelectrodes extend through the flexible polymeric substrate frombottom surface 18 to top surface 16 to define correspondingmicrolocations 14 on top surface 16 of substrate 12 (shown in FIG. 2Aand 2B). Details of APEX circuit 10 are more clearly shown in FIGS. 2Aand 2B. FIG. 2A is an enlarged top plan view of a portion of APEXcircuit 10 constructed on flexible polymeric substrate 12. APEX circuit10 has a plurality of microlocations 14 in polymeric substrate 12,comprising metal electrodes 15. FIG. 2B is an enlarged bottom plan viewof a portion of APEX circuit 10 depicted in FIG. 2A, showing bottomsurface 18 of flexible polymeric substrate 12, along with metal traces20 terminating in metal electrodes 15 located in microlocations 14. Inthe implementation depicted, each metal trace 20 connects to a differentmicrolocation 14 and includes a contact pad 21 (shown in FIG. 1). Inuse, metal traces 20 are in electrical contact with a voltage source byway of contact pad 21 that permits the creation of a biasingvoltage-across each of the electrodes. It will be appreciated that incertain implementations the biasing voltage at each electrode isindividually controllable and distinct from the other electrodes.However, in other implementations, the electrodes are controlled as oneor more groups.

FIG. 3 is a side cross-sectional view of APEX circuit 10 constructed onflexible polymeric substrate 12 shown in FIG. 2A and FIG. 2B.Hydrophilic permeation matrix 22 is positioned on the individualmicrolocation 14 and may extend onto the top surface 16 of the flexiblepolymeric substrate 12. In the embodiment depicted, metal trace 20 isdepicted on bottom surface 18 of flexible polymeric substrate 12.Although permeation matrix 22 is localized to the area of themicrolocation 14 corresponding to an electrode 15, it will beappreciated that in certain implementations permeation matrix 22 coversthe entire top surface 16 of polymeric substrate 12. It will also beappreciated that in other certain implementations permeation matrix 22is localized to the area of the microlocation 14 and may be of athickness different than that of the flexible substrate 12.

APEX circuit 10 may be manufactured using various methods and materials,according to the method disclosed in U. S. Pat. No. 5,401,913 (Columns3, 4, and 5, and FIGS. 1-7), the teachings of which are incorporatedherein by reference in their entirety. In reference now to FIGS. 4A, 4B,4C, 4D, and 4E, one implementation is depicted. As shown in FIG. 4A,metal layer 30 is deposited on a surface of flexible polymeric substrate32. Using conventional methodologies, metal layer 30 is subsequentlypatterned and etched in order to form a plurality of metallic traces 34,one of which is shown in cross-sectional view in FIG. 4B. After metaltraces 34 have been formed on flexible polymeric substrate 32, portionsof substrate 32 are removed or “milled” away from each of the electrodesto form vias 36 over each electrode 37, as shown in FIG. 4C, therebyexposing bare metal. Removal of substrate 32 can be performed by methodsknown in the art, including chemical, plasma, and laser processes. Inthe embodiment depicted, the bare metal of metal trace 34 has beenexpanded at the location of electrode 37 by deposition of additionalmetal to create protrusion 39. Protrusion 39 ensures better conductivityby increasing the surface area of electrode 37, and also provides forbetter adhesion of the metal trace 34 by forming lip 41 that slightlyoverlaps and interlocks with substrate 32. After via 36 has been formed,and protrusion 39 on electrode 37 created, hydrophilic matrix 38 isapplied, which coats electrode 37 and optionally fills via 36, as shownin FIG. 4E. Finally, appropriately defined microlocation 40 of matrix 38is doped with an appropriate receptor capable of determining a chemicalsignature of various test-species, as shown in FIG. 3E.

In reference now to FIG. 5, another configuration for an electrode in anAPEX array is depicted in cross-sectional partial view. In FIG. 5,microlocation 52 is first formed by depositing conducting metal 53 onflexible polymeric substrate 42. Suitable metals include aluminum, gold,silver, tin, copper, palladium, platinum, carbon and various metalcombinations. Special techniques for assuring the proper adhesion to theinsulating polymer substrate (e.g., polyimide or polyester) are usedwith different metals. The conducting metal is selectively removed toform a plurality of traces 44, one of which is shown in FIG. 5. Afterformation of trace 44, non-conducting masking layer 46 is applied overflexible polymeric substrate 42 such that it covers and substantiallyobscures metal trace 44. Non-conducting masking layer 46 is preferablyalso constructed of a flexible material that is capable of flexing alongwith polymeric substrate 42. Non-conducting masking layer 46 issubsequently milled and partially removed in order to reveal metal trace44, thereby forming electrode 48. After forming electrode 48, matrix 50is deposited over the top of masking layer 46. In the embodiment shown,matrix 50 entirely covers non-conducting masking layer 46. However, inother embodiments, matrix 50 may cover only a portion of masking layer46 or it may cover only the electrode 48. Thereafter portion 51 ofmatrix 50 is doped with, for example, receptor molecules atmicrolocation 52.

Yet another implementation of the invention is shown in FIG. 6, whichdepicts electrode 57 in which metal trace 54 has been enlarged to createraised area 56 projecting into via 58 formed in polymeric substrate 59.Electrode 57 is enlarged by deposit of metal. This deposit is formed,for example, by electroplating electrode 57 to selectively thicken thetop surface thereof. Alternatively, small metallic pieces may bemechanically inserted into each via above each electrode.57, and thesemetallic pieces can be solder re-flowed to create raised area 56. Suchthickening of electrode 57 can enhance performance of the APEX array bycreating a larger, more uniform electrode. In addition, raised area 56can enhance performance by effectively securing the metal layer to thesubstrate. After forming electrode 57, matrix 61 is deposited over thetop of substrate 59. In the embodiment shown, matrix 61 entirely coverssubstrate 59. However, in other embodiments, matrix 61 may cover only aportion of substrate 59 or only the electrode 57. Thereafter portion 63of matrix 61 is doped with, for example, receptor molecules.

FIG. 7 shows yet another implementation of the present invention, inwhich electroresistive layer 60 is shown positioned over electrode 62.FIG. 7 also shows flexible polymeric substrate 64, matrix 66, and matrixportion 68 that has been doped with, for example, receptor molecules. Inaddition, common ground plane 69 is placed over the top ofelectroresistive layer 60 and substrate 64. Ground plane 69 allowselectrical conduction from electrode 62, through resistive layer 60, andthen into ground plane 69. Due to the electrical resistance inelectroresistive layer 60, the temperature of gel portion 68 above theelectrode can be increased and controlled. If each of the electrodes inthe APEX circuit is individually controlled, the temperature of each ofthe electrodes can also be individually controlled. It will beappreciated that the resistive layer can be associated with theelectrode in different configurations, including positioning theelectrode between the resistive layer and the gel.

The embodiments depicted above show single-layer substrates with onelayer of metal traces. However, it will be appreciated that thesubstrate can have more than one layer. In addition, the metal tracesmay be positioned on one or more layers, depending upon the dictates ofthe application. For example, FIGS. 8A-8E show the steps of manufactureof yet another APEX circuit, and a cross-sectional view of a portion ofa finished APEX circuit. In FIG. 8A, flexible polymeric substrate 72 isshown with metal layer 74, depicted as two separate traces 74 a and 74b. Traces 74 a and 74 b may be formed from a single layer of metal byusing conventional etching methodologies. After forming separate traces74 a and 74 b, masking layer 76 is applied over the traces, as shown inFIG. 8B. Independent electrode 81 is formed by deposition of additionalmetal trace 84, as depicted in FIG. 8C. Thereafter, masking layer 76 andsubstrate 72 can be etched to form vias 78 and 82, after whichadditional metal may be electrodeposited to form electrodes 81 and 85 asshown in FIG. 8D. Vias 78 and 82 are subsequently filled withhydrophilic matrix 80, which may be, for example, an azlactone-bearingpolymer, as depicted in FIG. 8E. In implementations where high electrodedensity is desired, the metal traces are preferably layered in order toallow more electrodes to be positioned in a specific surface area.

In use, the array of microlocations 14 is brought into contact with asingle fluid volume that contacts top surface 16 but does not contactbottom surface 18. By applying electrical currents or voltage viacontact pads 21, free field electrophoresis can be controlled at eachmicrolocation 14. The single fluid volume can be a free-standing liquid(e.g., an aqueous buffer solution held in place by contact angle tohydrophobic flexible polymer substrate 12. Alternatively, as shown inFIG. 9, the single fluid volume can be defined by contacting top surface16 with fluid handling architecture 91 that is designed to confine aspecified volume of sample-containing fluid as single fluid volume 93over the array of microlocations 14. The enclosed volume over the arrayof microlocations 14 can be defined as APEX sample chamber 92. Fluidhandling architecture 91 can be designed to provide at least one inletport 94 and/or one outlet port 95 to allow a fluid sample to betransported into APEX sample chamber 92 for subsequent analysis.Optionally, fluid handling architecture 91 can provide for more than oneAPEX sample chamber 92 that can be mated to more than one array ofmicrolocations 14. Sample chambers thus formed can be fluidly connectedby inlet ports 96 and/or outlet ports 97 in the form of microchannels,microtubing, micropipettes, and the like.

In all of the embodiments, the flexible polymeric substrate ispreferably flexible enough such that it may be bent around a mandrel of2 feet in diameter without significant loss of integrity of theelectrical circuits. More preferably the flexible polymeric substratemay be bent around a mandrel 1 foot in diameter, and most preferablybent around a mandrel 6 inches in diameter, without significant loss ofintegrity of the circuits.

In certain implementations of the invention, the flexible polymericsubstrate is comprised completely, primarily, or partially of polyimide.Other flexible substrate materials may also be used, includingpoly(methylmethacrylate), polycarbonates, polyolefins, polyamides,polyvinyl chloride, and polytetrafluoroethylene, polyesters, or epoxies.Other ingredients which may be incorporated into the substrate includeplasticizers, toughening agents, pigments, fillers, stabilizers,antioxidants, flow agents, bodying agents, leveling agents, colorants,binders, fungicides, bactericides, surfactants, glass and ceramic beads,and reinforcing materials such as woven and non-woven webs of organicand inorganic fiber, provided that none of the added ingredientsinterfere with the chemical or biochemical processes for which the APEXarray is intended.

In certain implementations, the biologically receptive gel includes anazlactone-functional monomer. The gel advantageously is swellable,thereby providing an increased concentration of biologically receptivemolecules per unit area when used as a coating. The biologicallyreceptive gel is configured and arranged such that it may be patternedto a high resolution. This patterned biologically receptive gel may becured with actinic radiation, and the cured composition is capable ofreacting with selective biomolecules to immobilize the biomoleculesimmediately above the micro-electrodes. High-resolution patterning ofazlactone-functional gels, including gels containing biologically activematerials, is described in applicant's copending U. S. patentapplication Ser. No. 09/183197, filed Oct. 30, 1998, the teachings ofwhich are incorporated herein by reference in their entirety.

One preferred procedure for the derivitization of the metalmicro-electrode of the present invention uses aminopropyltriethoxysilane(APS) as a permeation and/or primer layer. APS reacts readily with theoxide and/or hydroxyl groups on metal surfaces to provide a high levelof functionalization, with only very limited binding to the remainingpolymer substrate. This primer will support the transport of small ionsand water as required to sustain electrophoresis, while impeding thediffusion of larger biomolecules. In addition, through a simple additionreaction to the APS primer film, the amine groups in the primer can bereacted with azlactone functional polymers to introduce a functionalhydrophilic matrix above the electrodes. This azlactone polymer can beconfigured with photocrosslinkers that enable the polymer to bephotolithographically patterned into gel pads at each microlocation.This azlactone functional gel contains additional functional groups thatcan be hydrolyzed to carboxylic acids. giving a hydrophilic gel. Theresulting patterned array of azlactone-functional gel pads, one on eachmicrolocation of the roll-good, can be reacted with biomolecules by asimple and quantitative addition reaction that does not requireco-reagents or removal of unwanted side products. Therefore, a roll-goodof the present invention can be doped with biomolecules by selectivelyprinting using a standard inkjet, flexographic, or other printingmethods. This overcomes the very tedious prior art process of usingfree-field electrophoresis to direct the covalently attachablebiomolecules to individual microlocations in preparation for covalentattachment.

The azlactone-functional monomer may be any suitable monomer containingan azlactone function group. A co-monomer may also be included, and theco-monomer may be any suitable monomer. Preferred monomers include vinylgroup-containing and acryl group-containing compounds. A representativelist of such monomers includes acrylamide, methacrylamide,N,-dimethylacrylamide, diacetoneacrylamide, N-vinylpyrrolidone,hydroxyethyl methacrylate, 2-acrylamido-2-methylpropanesulfonic acid andits salts, N-(3-methacrylidopropyl)-N,N,N-trimethylammonium salts,N,N-dimethylaminoethyl methacrylate, acrylic acid, methacrylic acid,itaconic acid, and combinations thereof. Preferred co-monomers areN,N-dimethylacrylamide and N-vinylpyrrolidone.

An example of a lithographically patternable azlactone functionalpolymer useful in the preparation of APEX chips is a copolymer preparedfrom equal amounts of vinyldimethylaziactone with dimethylacrylamide.This polymer composition can be reacted with a heterodifunctionalphotocrosslinker that has an amine functional group that binds to theazlactone, and an azido functional group that forms a crosslink toanother polymer chain upon application of UV light. This material isthermally reacted with the APS modified electrode surface to provide acovalent attachment to the electrode. The material is thenlithographically photocrosslinked to create individual gel pads at eachmicrolocation, and excess material is removed. Each gel pad has residualazlactone functionality that can then be hydrolyzed or selectivelyreacted with thiol- or amine-terminated biomolecules specific to thatmicrolocation.

Optional copolymeric crosslinkers may be included in the gel. Thesecrosslinkers may be any suitable species with two or more polymerizablefunctions. Suitable multifunctional crosslinking monomers includeethylenically unsaturated trimethylolpropane triacrylate andtrimethacrylate, (alpha-unsaturated) esters such as ethylene diacrylateand ethylene dimethacrylate, and amides, such asN,N′-dimethacryloyl-1,2-diaminoethane, and reaction products of2-alkenyl azlactones with short chain diamines.

The photocrosslinker may be any suitable chemical species that iscapable of binding two or more polymer molecules in response to theapplication of electromagnetic radiation and that is capable ofattaching to the gel-forming polymer at sites other than the end of agrowing polymer chain. The photocrosslinker should be capable ofattaching to the polymer after polymerization of the polymer iscomplete. Preferred photocrosslinkers include bisazides,bisdiazocarbonyls, and bisdiazirines. The bisazides are most preferredwith regard to ease of use. Azide (—N₃) groups release nitrogen (N₂)upon application of UV light, leaving behind highly reactive divalentnitrogen, i.e., nitrene. Reactive carbene can be generated by photolysisof a diazocarbonyl or a diazirine. Active nitrene or carbene species caninsert into many types of bonds including C—C or C—H bonds onpre-polymerized polymers to provide a crosslink. Azide, bisazide,azocarbonyl and bisdiazocarbonyl crosslinkers may formsubstrate-to-polymer gel links as well as polymer gel-to-polymer gelcrosslinks, and are therefore preferred. Preferred diazide speciesinclude 2,6-bis(4-azidobenzylidene)-4-methyl cyclohexanone (3AMC),4,4′diazidodiphenylether, 4,4′diazidodiphenylsulfone,4,4′diazidodiphenyl acetone, and 4,4′diazidodiphenylmethane. Preferredbisdiazocarbonyl species include 4,4-bis(m-diazobenzyl) benzene. Thephotocrosslinker may be capable of thermal cure as well as photocure.

In another implementation, the photocrosslinker may be ahetero-difunctional compound having a first function which attaches tothe polymer and a second function which forms crosslinks uponapplication of light. For example, 4[p-azidosalicyamido]butylamine(ASBA, available from Pierce Chemical Co., Rockford, Ill.) has an aminefunction that binds to an azlactone, and an azido function that forms acrosslink to another polymer chain upon application of UV light, asdescribed above.

In cases where the photocrosslinker attaches to the polymer by occupyingan azlactone function of the polymer, the crosslinker must be present inan amount less than 1 equivalent so that it does not occupy all of theazlactone sites necessary to bind biomolecules. In other words, thenumber of azlactone-reactive functions present on photocrosslinkers mustbe less than the total number of azlactone functions present on thepolymer. However, if the biomolecules are attached prior tophotocrosslinking, i.e., the composition already containsbiomolecule-azlactone bonds, then this condition need not apply.

The gel composition may be coated onto the flexible substrate, cured(i.e., photocrosslinked), patterned, and reacted with biomolecules. Byvarying methods, these steps may be done in any order. Many techniques,such as photolithographic patterning and laser induced thermal imaging(LITI) patterning, involve simultaneous or near-simultaneous stepsselected from coating, curing, or patterning of the composition. Apreferred approach to patterning gel pads onto the microlocations of theAPEX film is by LITI, because of the high registration accuracyachievable by this process. Specifically a high spot placement accuracyacross the web can be achieved for precisely transferring gel pads froma donor sheet onto the micro-locations on the web of APEX arrays.

Coating of the composition on the substrate may be achieved by anysuitable method. The coating may have a thickness in the range of 20 to500 μm, but is preferably between 0.05 and 100 μm and most preferablybetween 1 and 20 μm. The composition may be coated with or withoutaddition of solvent. Suitable methods include spin coating, spraycoating, knife coating, dipping, or roller coating. The composition maybe selectively coated to provide a patterned surface. Such methodsinclude known printing methods such as ink jet printing, offset,flexographic printing, etc. The composition may also be knife coatedonto a microstructured surface (e.g., a surface having micron scaledepressions or channels) in such a way that the composition resides onlyin the microstructures, providing a patterned array of the composition.

The gel may be cured by exposure to electromagnetic radiation,preferably UV light, most preferably UV-A light. The gel may beselectively cured by selective exposure to light. Selective exposuremethods include exposure through a mask or photographic negative orexposure by a directed beam of light or laser. Uncured gel may then beremoved, e.g., by washing, to provide a patterned coating. The gel maythen be further cured, either by light or heat cure. Heat cure mayentirely replace light cure in some circumstances, in particular wherethe gel is not patterned or is patterned by means other thanphotopatterning, such as by mechanical means.

Patterning may be achieved by a variety of means including selectivecoating of the gel on a substrate, selective curing of the gel, orselective removal of the gel from a substrate. It is an advantage of thepresent invention that the gel may be photopatterned to a resolution ofless than 2 micrometers by selective curing of the gel. Typical featuresare less than 1000 micrometers in size. Preferably, the patternedcoating has features of less than 200 μm in size. Preferably, thepatterned coating should cover the electrode surface 14. The sizedimensions referred to above are in-plane dimensions of the coatedfeatures or of the interstices between the coated features.

The doped gel may be patterned onto a substrate by laser addressablethermal transfer imaging (e.g., laser induced thermal imaging, or LITI)processes such as those described in U.S. Pat. No. 5,725,989,incorporated herein by reference. In this process, a thermal transferdonor element is constructed comprising a support layer, a light-to-heatconversion layer, and a transfer layer comprising the composition to bepatterned. When the donor element is brought in contact with a receptorand selectively irradiated to form a pattern or an image, a melt sticktransfer process occurs and the composition containing transfer layer isimaged onto the receptor. The photocrosslinkable azlactone compositionof this invention can be used in the transfer layer of such a system.This photocrosslinkable azlactone composition can be reacted withbiomolecules before incorporation into the transfer layer, afterincorporation into the transfer layer, or after laser addressed thermaltransfer to the receptor. The azlactone composition of this inventioncan be thermally or photochemically crosslinked before or after thetransfer process.

This process offers the opportunity to pre-pattern differentbiomolecules onto a transfer layer comprising the present azlactonecomposition prior to laser addressed thermal imaging of individualazlactone-biomolecule conjugates to the receptor substrate. Registrationof the elements may be robotically altered between any or all of thetransfer steps to build up desired array spacing and size fortransferred elements on the receptor that is different from thepatterning of the biomolecules on the transfer layer. The laseraddressable thermal imaging process offers high resolution imaging andhigh registration accuracy.

The azlactone functions of the doped polymer may bind to a variety ofattaching functions present on biomolecules, including, primary amine,secondary amine, hydroxy and thiol. These groups react, either in thepresence or absence of suitable catalysts, with azlactones bynucleophilic addition to produce a residue of a biomolecule bound to aresidue of an azlactone function.

Depending on the attaching function of the biomolecule, catalysts may berequired to achieve effective attaching reaction rates. Primary amine orthiol functions require no catalysts. Acid catalysts such astrifluoroacetic acid, ethanesulfonic acid, toluenesulfonic acid, and thelike are effective with hydroxy and secondary amine functions. The levelof catalyst employed is generally from 1 to 10 parts, preferably 1 to 5parts, based on 100 parts of azlactone. The azlactone functions of thepresent invention advantageously prefer attachment to carbohydrated,polypeptide and/or polynucleotide sequences at a terminal end.

The step of attaching biomolecules to the polymer may be carried outbefore or after coating, before or after curing, and before or afterpatterning. After biomolecules are attached, capping groups may be addedto occupy any unused azlactone functions and prevent later contaminationof the gel by any unwanted material. Capping groups preferably reactreadily with the azlactone and preferably do not interfere with thedesired characteristics of the gel. Preferably, the capping group iswater soluble and thus improves the swellability of the gel.

The resulting cured gel is advantageously swellable, since a swellablegel may bind a greater amount to the biomolecule in a given area of asubstrate. This increase in a real density improves the ability to takemeasurements such as optical readings of the target site and allows forgreater miniaturization. This invention is useful in analytical devicesemploying biomolecules as test probes or reference standards, inparticular where it is desirable to employ a variety of differingbiomolecules in a compact area of a substrate. For example,miniaturization of DNA sequencing operations onto microchips offersadvantages in speed and cost. Oligonucleotide containing gel pads of thepresent invention can be used to make high density DNA chips havinganywhere from thousands to millions of microelectrodes spotted on thesurface of a chip. In the method of sequencing by hybridization, acomplete array of oligonucleotide probes (e.g., all 1024 possiblepentamers or all 65,536 possible octamers) is patterned onto a substrateand the DNA sample to be sequenced is allowed to specifically hybridizeto the array. The hybridization process can take several hours ifgoverned by simple diffusion of the DNA molecules. By applying currentor voltage signals to the microlocations of the present invention, DNAsamples can be quickly attracted to the individual gel pads forhybridization. Reversing the signal drives off non-specifically boundsample. Repeating the process several times can enhance sample uptake atthe correctly specified microlocations on the APEX array. In thisapplication, electrodes can be individually addressed or can beaddressed in parallel. In the later case, the electrodes can optionallybe connected to a common ground plane. The target DNA sequence can beidentified by analysis of the overlapping sets of oligomers that formperfect duplexes with the target sequence. As an example, chips such asthese may be useful in applications that require sequencing of multiplegene mutations, as might be required in detecting a polygenic disease.

Low density DNA chips generally can have up to 300 probes and areparticularly suited to diagnostic applications where detection of aspecific organism or strain or a panel of tests is required. A sample ofDNA can be electrophoretically moved from one microlocation to the next.Electronic stringency control can be used to retain the DNA that matchesthe capture probe at each microlocation.

Microarrays of enzyme-containing gel pads of the present invention canbe used to screen chemical compounds for enzyme inhibiting or activatingeffect. Biomolecules on the surface of microorganisms serve as anchorpoints for attachment to the gel. The biological response of themicroorganisms to chemical stimuli or other environmental conditions canbe monitored. Gels containing biological molecules that promote cellattachment and growth (e.g., growth factors or collagen) can bepatterned using the methods and compositions describe above. Theresultant patterned gels can be used to generate two-dimensionalcellular structures.

Also, the individual gel pads may be used as microreactors. For example,polymerase enzyme may be covalently anchored to individualmicroelectrodes and used to support DNA amplification at specifiedmicrolocations. In this case, the electrodes are charged to introducereagents and transfer amplified products to specified microlocations forfurther processing.

Devices of the present invention can also be used to support immunoassaypanels, panels for drugs of abuse, enzyme based electrodes and optodes.Also, an array of gel pads can be used to soak up a metered amount offluid containing microorganisms to be detected. Growth nutrients andfluorescent probes can be incorporated in the gel pads. The number ofviable organisms in the sample can be related to the number of gel padsthat exhibit a fluorescent response.

In addition, devices of the present invention can be used to screenlibraries of carbohydrate oligomers for specific affinities tobiomolecules such as drug receptors. Carbohydrate oligomers terminatedwith an amino- or thio-linkage can be covalently attached toazlactone-functional microlocations on the APEX array. A samplecontaining a specific receptor can be electrophoretically concentratedonto one or more microlocations during the assay. Electronic stringencycontrol can be used to determine relative affinities for the receptor toindividual carbohydrate oligomers in the assay.

Flexible APEX arrays of the present invention can be laminated to astructured fluid handling architecture (glass or plastic) designed todirect or confine a fluid to the region above the array ofmicro-locations on the first surface of the substrate. In a preferredembodiment, a microreplicated plastic film may be laminated to the firstsurface of a flexible APEX array film such that the resulting laminatemay define a series of APEX chips which is can be diced into individualdevices or stored as-a roll good for future use. In this preferredembodiment, the electrode traces and contact pads can be patterned onthe second surface of the APEX film, such that the contact padsterminate at the edge of each individual chip, once diced. Themicroreplicated film preferably can be embossed with features that, whenlaminated, define a closed sample well above the array of APEXmicrolocations. Typical dimensions for such a well may be 1 cm×1 cm by50 μm deep. At least two microreplicated channels may connect this wellto sample inlet and outlet ports defined by vias in the microreplicatedfilm. The film may define additional microfluidic processingarchitectures, as needed.

In use, this chip can be inserted into an APEX controller designed tomate to the inlet and outlet ports as well as the contact pads. Thecontroller can inject a sample onto the chip and optically interrogatethe APEX array while actively processing the sample through applicationof controlled voltages or controlled currents to the individualmicroelectrodes.

In another embodiment, as shown in FIGS. 10a and 10 b, APEX biocard 100can be prepared by laminating a sheet of APEX film 190, having between 2and 200 separate APEX arrays 110, shown in FIG. 10A, to a rigid glass orpreferably a micromolded plastic fluid-handling architecture 120, inregistration with the APEX arrays, to form biocard 100, shown in FIG.10b. In this embodiment, fluid handling architecture 120 can be designedto define between 2 and 200 independent samples wells 130, one for eachAPEX array 110, with corresponding inlet channels 140 and outletchannels 150. In this format, between 2 and 200 different biologicalsamples, corresponding to the number of APEX arrays 110 on biocard 100,can each be evaluated. The fluid handling architecture can be simplebarriers between APEX arrays (e.g., open wells into which sample isinjected, such that the cassette must be horizontal), to closedstructures into which sample can be injected with a syringe or pump,such that the cassette can be held in a vertical position.

A machine may also be provided for accepting and operating an APEXbiocard or cassette. The APEX biocard controller can comprise a sampleinjection unit to provide 2 to 200 independent biological samples ontothe biocard via ports in the fluid handling architecture. A voltagecontrol unit can simultaneously provide controlled currents or voltagesto each of the 2 to 200 APEX arrays in parallel, for electrophoreticprocessing. A detection system can provide optical, electrical, ormechanical signals in response to biological events at the individualelectrodes of each APEX array.

In one embodiment, the intake ports may be configured to receive fluidinjection tips and related assembly, through which samples arrive at theAPEX arrays, under a vacuum applied to the biocard, which is thenreleased to atmospheric pressure. The injection port optionally mayinclude a small intake reservoir, which acts as a fluid buffer. Thefluid (e.g., patient sample or other solution) can enter the intakeport, collect in the intake reservoir, and travel along a distributionchannel to the sample well. Each fill channel can descend to and enterthe sample well at an angle which results in a natural flow of thesample fluid down through the fill channel by gravity. Each of thesample wells may have an associated bubble trap, connected to the samplewell and located at a height slightly above the well on the cardsurface. Each bubble trap can be connected to its respective well by ashort conduit.

In operation, each of 2 to 200 samples can be processed simultaneouslywith one controller unit that operates the bio-card. A single controlledvoltage or current source can apply control signals to all of the APEXarrays in parallel. An imaging system mounted on an X-Y stage, movingfrom one array to the next, can collect optical information about theelectrophoretically processed sample on each array.

In yet another embodiment, as shown in FIG. 11, a spool containing roll210 of APEX film to which a flexible plastic fluid handling architecturehas been laminated as described above, is prepared. The fluid handlingarchitecture can be designed to direct 2-10,000 independent biologicalsamples to the corresponding number of independently addressable APEXarrays on APEX film 220 for further processing. Film 220 can be advancedthrough assay machine 230 that addresses a preselected subset of APEXarrays. Once the samples have been assayed, the spool can be furtheradvanced.

Assay machine 230 may include a sample injection unit to providebiological samples onto the cassette via ports in the fluid handlingarchitecture. A voltage control unit may simultaneously provideprocessing currents or voltages to each of the APEX arrays that may beresident in assay machine 230. A detection system can provide optical,electrical, or mechanical signals in response to biological events atthe individual electrodes of each APEX array.

The invention is further described by the following example:

EXAMPLE

Free Field Electrophoresis on a Microcircuit

A laminated microinterconnect (LMI) circuit essentially identical tothat shown in FIG. 1 was prepared according to the method disclosed inU. S. Pat. No. 5,401,913 (Columns 3, 4, and 5, and FIGS. 1-7),comprising a polyimide substrate having copper microcircuit traces onone side. Vias of approximately 60 μm diameter were chemically milledfrom the side opposite the microcircuits in registration with the coppercircuit termini, on a 200 μm pitch.

An aqueous solution containing bovine serum albumin (BSA, Sigma ChemicalCo., St. Louis, Mo.) that was labeled with fluorescein isothiocyanate(FITC) in a pH 7.0 phosphate buffered saline buffer (PBS) was applied tothe LMI on the side having vias. Electrical connection was made toindividual electrodes using platinum microprobes and voltages wereapplied using a computer-controlled voltage supply. At pH 7.0, the BSAwas positively charged and accumulated at negatively biased electrodes.Using a Leica epifluorescence microscope equipped with an L3 filter cube(Leica Microsystems, Inc., Deerfield, Ill.), fluorescence of labeled BSAcould be detected at vias where it had accumulated according to thenegative bias of the electrodes. Fluorescence was induced by irradiationat 470 nm using a blue LED and monitored at 510 nm by the Leicamicroscope.

Thus, FITC-labeled BSA was electrophoretically concentrated onto one ofthe electrodes by applying a 10 Volt bias to that electrode while allother electrodes were grounded. A second electrode within the microscopefield of view was then biased at −10 Volts while the first electrode wasgrounded. Within a few seconds, the labeled BSA was seen to migrate tothe second negative electrode. Repeated cycling of charge wasaccompanied by repeated migration of the labeled BSA.

The above specification and example are believed to provide a completedescription of the manufacture and use of particular embodiments of theinvention. Many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention.

We claim:
 1. An electronic device adapted for performing electrophoresisassisted processes, the device comprising: at least one flexiblepolymeric substrate having a first surface and a second surface; one ormore microlocations interrupting the first surface, each of saidmicrolocations including an electrode disposed on the second surface ofthe flexible substrate; and a hydrophilic matrix positioned on the firstsurface of the flexible substrate and in electrical contact with atleast one of the electrodes, further comprising a plurality ofconductive traces, each trace connected to at least one of saidelectrodes, wherein said device is capable of bending around a mandrelhaving a diameter of 24 inches while maintaining the integrity of saidconductive traces.
 2. An electronic device adapted for performingelectrophoresis assisted processes, the device comprising; at least oneflexible polymeric substrate having a first major surface; one or moremicrolocations disposed on the first major surface of the substrate,each of the microlocations including an electrode; and a hydrophilicmatrix positioned on the first major surface of the substrate and inelectrical contact with at least one of the electrodes disposed thereon,wherein said device is capable of bending around a mandrel having adiameter of 12 inches while maintaining the integrity of said conductivetraces.
 3. An electronic device adapted for performing electrophoresisassisted processes, the device comprising; at least one flexiblepolymeric substrate having a first major surface; one or moremicrolocations disposed on the first major surface of the substrate,each of the microlocations including an electrode; and a hydrophilicmatrix positioned on the first major surface of the substrate and inelectrical contact with at least one of the electrodes disposed thereon,wherein said device is capable of bending around a mandrel having adiameter of 6 inches while maintaining the integrity of said conductivetraces.
 4. A method of making an electronic device adapted forperforming molecular biological processes, the method comprising:providing a flexible polymeric substrate having a first surface and asecond surface; forming a plurality of microlocations interrupting thefirst surface, each of said microlocations including an electrodedisposed on the second surface of the flexible substrate; and applying ahydrophilic matrix on at least one of the first surface of the flexiblesubstrate and the microlocations, wherein the hydrophilic matrix makeselectrical contact with the electrode, further comprising the step ofapplying at least one of a biological material and a chemical materialto at least one of the microlocations, wherein the hydrophilic matrix isapplied to said at least one microlocation by laser addressable thermaltransfer imaging.
 5. The method according to claim 4 wherein thehydrophilic matrix further comprises a biologically receptive polymer.6. A method of making an electronic device adapted for performingmolecular biological processes, the method comprising: providing aflexible polymeric substrate having a first surface and a secondsurface; forming a plurality of microlocations interrupting the firstsurface, each of said microlocations including an electrode disposed onthe second surface of the flexible substrate; and applying a hydrophilicmatrix on at least one of the first surface of the flexible substrateand the microlocations, wherein the hydrophilic matrix makes electricalcontact with the electrode, further comprising the step of applying atleast one of a biological material and a chemical material to at leastone of the microlocations, wherein said at least one of a biologicalmaterial and a chemical material is applied to the at least onemicrolocation by photolithographic imaging.
 7. An electronic deviceadapted for performing electrophoresis assisted processes, the devicecomprising: a flexible polymeric substrate having a first surface and asecond surface, the substrate being at least partially wrapped around aspool; a plurality of microlocations interrupting the first surface ofthe substrate, each of said microlocations including an electrodedisposed on the second surface of the flexible substrate; and ahydrophilic matrix positioned on the first surface of the flexiblesubstrate and in electrical contact with the electrode.
 8. Theelectronic device according to claim 7, wherein the hydrophilic matrixcomprises a biologically receptive gel.
 9. The electronic deviceaccording to claim 7, wherein the flexible polymeric substrate comprisespolyimide.
 10. The electronic device according to claim 7, wherein theplurality of microlocations interrupting the first surface comprise viasthat extend from the first surface into the polymeric substrate.
 11. Theelectronic device according to claim 7, wherein the plurality ofmicrolocations interrupting the first surface further comprise addedconductive material.
 12. The electronic device according to claim 7,wherein the microlocations have a smallest dimension in the plane of thefirst surface of less than 200 μm.
 13. A device for processing aplurality of APEX devices disposed on a flexible polymeric substrate inreel format, the device comprising: a plurality of APEX devices disposedon a flexible polymeric substrate in reel format, each APEX devicecomprising a flexible polymeric substrate having a first surface; ahydrophilic matrix positioned on the first surface of the flexiblepolymeric substrate; a plurality of microlocations interrupting thefirst surface, each of said microlocations including an electrodedisposed on the second surface of the flexible substrate and inelectrical contact with the electrode and a fluid handling architecture;at least one sample injection means; a voltage control means; and adetection system to provide one of optical, electrical and mechanicalsignals in response to biological events at least one of the electrodesof the APEX arrays.